Flow cell devices and optical systems for in situ nucleic acid sequencing

Abstract

Fluorescence imaging systems designs, flow cell devices, and methods of are described herein that enable imaging of three or more axially displaced surfaces without using any optical compensators. The optical systems and flow cell devices herein provides higher throughput analysis for genomics and other imaging applications at a lower cost.

What is claimed is: 
     
         1 . A system for in situ biomolecule analysis, the system comprising:
 an imaging system comprising:
 a flow cell configured to hold a cell or a tissue immobilized thereon, wherein said cell or said tissue comprises a plurality of analytes that differ in type from each other; 
 a light source configured to illuminate said cell or said tissue, thereby generating a plurality of signals corresponding to the plurality of analytes; and 
 a detector configured to image said plurality of signals; and 
 one or more processors communicatively coupled to said imaging system, wherein said one or more processors is individually or collectively programed to
 (a) illuminate, using said light source, said cell or said tissue, thereby generating said plurality of signals corresponding to said plurality of analytes; 
 (b) detect, using said detector, said plurality of signals; and 
 (c) determine, using said one or more computer processors, an identity or sequence of said plurality of analytes using said plurality of signals. 
 
   
     
     
         2 . The system of  claim 1 , wherein said cell or tissue is an in situ cell or tissue sample. 
     
     
         3 . The system of  claim 1 , wherein said light source is configured to illuminate greater than about 20 square millimeters (mm 2 ) of said flow cell and said cell or said tissue with a peak-to-valley energy or power variation of at most about 5%. 
     
     
         4 . The system of  claim 1 , wherein said light source is configured to illuminate greater than about mm 2  of said flow cell and said cell or said tissue with a RMS wavefront error of at most about 0.092. 
     
     
         5 . The system of  claim 1 , wherein said imaging system has a composite root mean square error of less than about 0.05. 
     
     
         6 . The system of  claim 1 , wherein said cell or said tissue is a whole cell or whole tissue. 
     
     
         7 . The system of  claim 1 , wherein said imaging system does not comprise an objective disposed within an optical path of said light source or said detector. 
     
     
         8 . The system of  claim 7 , wherein said imaging system does not comprise an objective. 
     
     
         9 . The system of  claim 1 , wherein said imaging system does not comprise a tube lens. 
     
     
         10 . The system of  claim 1 , wherein said illumination has an irradiance of at least about 40 milliwatts per square meter. 
     
     
         11 . The system of  claim 1 , wherein said cell or said tissue has been permeabilized. 
     
     
         12 . The system of  claim 1 , wherein said plurality of signals are a plurality of fluorescent signals. 
     
     
         13 . The system of  claim 1 , wherein said plurality of signals are detected with a Q-score of at least 30, 40, or 50. 
     
     
         14 . The system of  claim 1 , wherein said cell or said tissue is illuminated with 10 illumination fields in one or more planes perpendicular to an optical axis of said imaging system. 
     
     
         15 . The system of  claim 1 , wherein a field of view of said detector is at least about 10 mm 2 . 
     
     
         16 . The system of  claim 1 , wherein said cell or said tissue is imaged with a resolution of at least about 1 micrometer. 
     
     
         17 . The system of  claim 1 , wherein said flow cell is configured to permit the flow of one or more reagents into contact with said cell or said tissue. 
     
     
         18 . The system of  claim 1 , wherein said cell or said tissue is a cultured cell or a cultured tissue. 
     
     
         19 . The system of  claim 1 , wherein said cell or said tissue is an isolated cell or an isolated tissue. 
     
     
         20 . The system of  claim 1 , wherein a fidelity of imaging of said cell or said tissue is at least about 0.1 micrometers. 
     
     
         21 . The system of  claim 1 , wherein said plurality of analytes comprise a nucleic acid molecule. 
     
     
         22 . The system of  claim 21 , wherein said nucleic acid molecule is a deoxyribonucleic acid molecule. 
     
     
         23 . The system of  claim 21 , wherein said nucleic acid molecule is a ribonucleic acid molecule. 
     
     
         24 . The system of  claim 1 , wherein said plurality of analytes comprise a protein. 
     
     
         25 . The system of  claim 1 , wherein said plurality of analytes comprise a carbohydrate. 
     
     
         26 . A method for imaging an in situ sample, comprising:
 (a) providing said in situ sample comprising a plurality of different types of analytes;   (b) illuminating said plurality of different types of analytes to generate a plurality of signals related to said plurality of analytes; and   (c) imaging said plurality of signals.   
     
     
         27 . The method of  claim 26 , wherein said illuminating said plurality of different types of analytes is a sequential illumination of said plurality of different types of analytes. 
     
     
         28 . The method of  claim 26 , wherein said illuminating said plurality of different types of analytes is a simultaneous illumination of said plurality of different types of analytes. 
     
     
         29 . The method of  claim 26 , wherein said plurality of different types of analytes are selected from the group consisting of deoxyribonucleic acid molecules, ribonucleic acid molecules, proteins, morphological features, and phosphorylated proteins. 
     
     
         30 . The method of  claim 26 , further comprising applying one or more sequencing reagents on said in situ sample configured to sequence said plurality of different types of analytes. 
     
     
         31 . The method of  claim 26 , wherein said illuminating is over an area of said flow cell that is greater than about 20 square millimeters (mm 2 ) has a peak-to-valley variation of at most about 5%. 
     
     
         32 . The method of  claim 26 , wherein said illuminating is over at least about 1 mm 2  of said flow cell with a RMS wavefront error of at most about 0.092. 
     
     
         33 . The method of  claim 26 , wherein said illuminating is over an area of said flow cell that is greater than about 20 square millimeters (mm 2 ) has a peak-to-valley variation of at most about 5%. 
     
     
         34 . The method of  claim 26 , further comprising (d) using a computer processor operatively coupled to said detector to analyze said plurality of signals. 
     
     
         35 . The method of  claim 34 , wherein said analyzing said plurality of signals comprises determining a sequence of a nucleic acid molecule within said in situ sample. 
     
     
         36 . The method of  claim 35 , wherein said sequence of said nucleic acid molecule is determined with an accuracy, sensitivity, or specific of at least about 95%. 
     
     
         37 . The method of  claim 36 , wherein said sequence of said nucleic acid molecule is determined in an absence of altering a spatial relationship of said nucleic acid within said in situ sample. 
     
     
         38 . The method of  claim 26 , wherein said in situ sample has a length, width, or height of at least about 10 micrometers. 
     
     
         39 . The method of  claim 26 , wherein said in situ sample comprises a tissue. 
     
     
         40 . The method of  claim 26 , wherein said in situ sample comprises a plurality of cultured cells. 
     
     
         41 . The method of  claim 26 , wherein said in situ sample comprises a plurality of isolated cells. 
     
     
         42 . The method of  claim 26 , wherein said in situ sample is imaged with about 10 images in a plane perpendicular to an optical axis of said optical assembly. 
     
     
         43 . The method of  claim 26 , wherein said plurality of signals are a plurality of fluorescent signals. 
     
     
         44 . The method of  claim 26 , wherein said plurality of signals are detected with a Q-score of at least 30, 40, or 50. 
     
     
         45 . The method of  claim 26 , wherein said in situ sample comprises a nucleic acid molecule. 
     
     
         46 . The method of  claim 45 , wherein said nucleic acid molecule is a deoxyribonucleic acid molecule. 
     
     
         47 . The method of  claim 45 , wherein said nucleic acid molecule is a ribonucleic acid molecule. 
     
     
         48 . The method of  claim 26 , wherein a field of view of said optical assembly is at least about 10 mm 2 . 
     
     
         49 . The method of  claim 26 , wherein said in situ sample is imaged at a resolution of at least about 1 micrometer. 
     
     
         50 . The method of  claim 26 , wherein said in situ sample is imaged within at most about 24 hours. 
     
     
         51 . The method of  claim 26 , wherein a fidelity of imaging a plurality of images of said in situ sample is at least about 0.1 micrometers. 
     
     
         52 . An optical assembly for in situ imaging, comprising:
 a flow cell configured to contain an in situ sample;   a light source configured to illuminate said in situ sample in said flow cell, thereby generating a signal related to a property of said in situ sample; and   a detector configured to image said signal.   
     
     
         53 . The optical assembly of  claim 52 , wherein said illumination over an area of said flow cell that is greater than about 20 square millimeters (mm 2 ) has a peak-to-valley energy or power variation of at most about 5%. 
     
     
         54 . The optical assembly of  claim 52 , wherein said illumination has a root-mean-square (RMS) wavefront error of at most about 0.092 over an area of at least about 1 square millimeter (mm 2 ). 
     
     
         55 . The optical assembly of  claim 52 , further comprising a processor configured to analyze said signal to determine said property of said in situ sample. 
     
     
         56 . The optical assembly of  claim 52 , wherein said in situ sample has a length, width, or height of at least about 10 micrometers. 
     
     
         57 . The optical assembly of  claim 52 , wherein said optical assembly does not comprise an objective. 
     
     
         58 . The optical assembly of  claim 57 , wherein said system does not comprise an objective. 
     
     
         59 . The optical assembly of  claim 52 , wherein said optical assembly does not comprise a tube lens. 
     
     
         60 . The optical assembly of  claim 59 , wherein said system does not comprise an objective. 
     
     
         61 . The optical assembly of  claim 52 , wherein said in situ sample comprises a tissue. 
     
     
         62 . The optical assembly of  claim 52 , wherein said in situ sample comprises a plurality of cultured cells. 
     
     
         63 . The optical assembly of  claim 52 , wherein said in situ sample comprises a plurality of isolated cells. 
     
     
         64 . The optical assembly of  claim 52 , wherein said in situ sample is imaged with at most about 10 images in a plane perpendicular to an optical axis of said optical assembly. 
     
     
         65 . The optical assembly of  claim 52 , wherein said signal is a fluorescent signal. 
     
     
         66 . The optical assembly of  claim 52 , wherein said signal is detected with a Q-score of at least about 30. 
     
     
         67 . The optical assembly of  claim 52 , wherein said in situ sample comprises a nucleic acid molecule. 
     
     
         68 . The optical assembly of  claim 67 , wherein said nucleic acid molecule is a deoxyribonucleic acid molecule. 
     
     
         69 . The optical assembly of  claim 67 , wherein said nucleic acid molecule is a ribonucleic acid molecule. 
     
     
         70 . The optical assembly of  claim 52 , wherein a field of view of said optical assembly is at least about 10 mm 2 . 
     
     
         71 . The optical assembly of  claim 52 , wherein said in situ sample is imaged at a resolution of at least about 1 micrometer. 
     
     
         72 . The optical assembly of  claim 52 , wherein said in situ sample is imaged within at most about 24 hours. 
     
     
         73 . The optical assembly of  claim 52 , wherein a fidelity of imaging a plurality of images of said in situ sample is at least about 0.1 micrometers. 
     
     
         74 . A method for imaging an in situ sample, comprising:
 (a) providing said in situ sample in a flow cell comprised within a system comprising an optical assembly comprising a light source and a detector;   (b) illuminating said in situ sample and generating a signal related to an analyte of said in situ sample; and   (c) imaging, using said detector, said signal.   
     
     
         75 . The method of  claim 74 , wherein said illuminating is over an area of said flow cell that is greater than about 20 square millimeters (mm 2 ) has a peak-to-valley variation of at most about 5%. 
     
     
         76 . The method of  claim 74 , wherein said illuminating is over at least about 1 mm 2  of said flow cell with a RMS wavefront error of at most about 0.092. 
     
     
         77 . The method of  claim 74 , further comprising (d) using a computer processor operatively coupled to said detector to analyze said signal. 
     
     
         78 . The method of  claim 77 , wherein said analyzing said signal comprises determining a sequence of a nucleic acid molecule within said in situ sample. 
     
     
         79 . The method of  claim 78 , wherein said sequence of said nucleic acid molecule is determined with an accuracy, sensitivity, or specific of at least about 95%. 
     
     
         80 . The method of  claim 79 , wherein said sequence of said nucleic acid molecule is determined in an absence of destroying said in situ sample. 
     
     
         81 . The method of  claim 74 , wherein said in situ sample has a length, width, or height of at least about 10 micrometers. 
     
     
         82 . The method of  claim 74 , wherein said optical assembly does not comprise an objective. 
     
     
         83 . The method of  claim 82 , wherein said system does not comprise an objective. 
     
     
         84 . The method of  claim 74 , wherein said optical assembly does not comprise a tube lens. 
     
     
         85 . The method of  claim 84 , wherein said system does not comprise an objective. 
     
     
         86 . The method of  claim 74 , wherein said in situ sample comprises a tissue. 
     
     
         87 . The method of  claim 74 , wherein said in situ sample comprises a plurality of cultured cells. 
     
     
         88 . The method of  claim 74 , wherein said in situ sample comprises a plurality of isolated cells. 
     
     
         89 . The method of  claim 74 , wherein said in situ sample is imaged with at most about 10 images in a plane perpendicular to an optical axis of said optical assembly. 
     
     
         90 . The method of  claim 74 , wherein said signal is a fluorescent signal. 
     
     
         91 . The method of  claim 74 , wherein said signal is detected with a Q-score of at least about 30. 
     
     
         92 . The method of  claim 74 , wherein said in situ sample comprises a nucleic acid molecule. 
     
     
         93 . The method of  claim 92 , wherein said nucleic acid molecule is a deoxyribonucleic acid molecule. 
     
     
         94 . The method of  claim 92 , wherein said nucleic acid molecule is a ribonucleic acid molecule. 
     
     
         95 . The method of  claim 74 , wherein a field of view of said optical assembly is at least about 10 mm 2 . 
     
     
         96 . The method of  claim 74 , wherein said in situ sample is imaged at a resolution of at least about 1 micrometer. 
     
     
         97 . The method of  claim 74 , wherein said in situ sample is imaged within at most about 24 hours. 
     
     
         98 . The method of  claim 74 , wherein a fidelity of imaging a plurality of images of said in situ sample is at least about 0.1 micrometers.